Plant Growth Regulators and Biotechnology

Introduction

A plant growth regulator is an organic compound, either natural or synthetic, that modifies or controls one or more specific physiological processes within a plant. If the compound is produced within the plant it is called a plant hormone. A plant regulator is defined by the Environmental Protection Agency as "any substance or mixture of substances intended, through physiological action, to accelerate or retard the rate of growth or maturation, or otherwise alter the behavior of plants or their produce. Additionally, plant regulators are characterized by their low rates of application; high application rates of the same compounds often are considered herbicidal". Upon reading these descriptions, it strikes me that the definition is likely to be broadened, particularly as it relates to the EPA, as we consider changes that can be made through the modern methods of genetic engineering.

Exactly how do changes made through genetic engineering compare to classical methods of genetic manipulation? An example that contrasts the two approaches is helpful in understanding the differences and similarities between these two methods. Both classical and molecular approaches were used to increase the sugar content of the commercially available tomato. This work was made possible because certain wild tomato relatives, although unlike commercial varieties in appearance, have a higher sugar content. The plan was to transfer the higher sugar content of the wild tomato to the domesticated tomato and leave behind its smaller size, lower yield and bitter taste.

The classical breeding approach accomplished this goal through the mixing of genetic information from the two parents upon sexual exchange. After many cycles of backcrossing to the parental commercial variety, most information from the wild parent was eliminated but still some remained. In that uncharacterized bit of genetic material was the information for the sweetness characteristic, but also the information for an unexpected characteristic. The newly developed variety had lowered fertility. This is because the breeder did not have total control over exactly what information was retained in the new tomatoes. They tried to minimize the unwanted information from the wild species, but despite this effort they were unable to eliminate everything but the sweetness characteristic. They ended up with a new variety that still had a substantial input from the wild species and in that information was the sweetness characteristic and something that caused lower fertility.

In the genetic engineering approach, the researchers found a single piece of genetic information that when removed would slow the breakdown of sugar. Through genetic engineering technologies, they were able to build that characteristic into the commercial variety and stop sugar degradation in the commercial tomato. They did this by making a very specific change in a single gene. They changed nothing else about the tomato. Additionally, in contrast to classical breeding, they knew precisely what information they were adding. Another difference with this approach is that, although in this case the changed information came from the tomato species itself, it would have been possible to take that new genetic information from another plant species, a microbe or any other organism for that matter. In addition with this approach it is possible to "define" precisely when and where this newly acquired information will be made by using characterized regulatory regions that function when and where they are need

How is Biotechnology impacting agriculture?

What is being done with the new technology of biotechnology in the arena of plants. Today, there are products in the field and the marketplace. The new technologies of genetic engineering can result in more environmentally friendly plant pest-protection, foods with enhanced nutrition, more accurate and sensitive diagnostics, foods with improved processing and marketing characteristics, better and more efficient medical delivery, new methods for removing contaminants from soils and waters, and creation of products that are presently being made from nonrenewable resources. Many of these changes might be considered under the broad definition of a "plant growth regulator".

While products of this technology once were confined to the research laboratory, this is no longer the case. These and other new crops represent a substantial percentage of actual production acreage in the U.S. In 1998 50% of the cotton acreage, 30% of the soybean and 20% of the maize acreage in the U.S. were genetically engineered. In the U.S. some 60 million acres of G.E. crops were planted in 1998 and the projections for 1999 are that this number will increase, with a wider variety of products available to farmers and present in the marketplace..

In the short term and long term, what might some of these products be?

Pest Control

Herbicide tolerance. A major agricultural advance occurred with the identification of selective herbicides that can be used to eliminate weeds without affecting the crop plant. But achieving complete selectivity has been problematic for many crops. An alternative to finding new selective herbicides is to genetically engineer the crop to resist existing herbicides. The engineered mechanisms can include modifications in the site of action of the herbicide, in its detoxification or in the uptake and translocation of the herbicide to its site of action. Such crops represent the first and most frequent application of biotechnology to agriculture. Of the total of 5900 permits and notifications issued through the end of 1998 by the USDA Animal and Plant Health Inspection Service (APHIS), 27% or 1569 permits were for herbicide-tolerant crops (http//:www.nbiap.vt.edu/cfdocs/biocharts2cfm). Based on data from APHIS, work is currently being done on six herbicides: bromoxynil, glyphosate, imadazolinones, phosphinothricin

In many cases, the herbicide tolerance is based on a modified site of action through the use of mutant or altered forms of the target protein. In these instances, the altered enzyme must be properly compartmentalized (chloroplast, cytoplasm, nucleus) and it must be assembled in a metabolically active form at its target location. This is the case with glyphosate where the herbicide blocks a pathway that leads to the synthesis of several amino acids; blockage of this pathway leads to starvation for amino acids. Genetic engineering strategies have focused mainly on using mutant target enzymes that are resistant to glyphosate. A similar type of strategy is used with another group of herbicides, the imadazolinones and sulfonylureas, which also inhibit a key step in the biosynthesis of a different set of amino acids.

Other approaches to engineering herbicide resistance involve the introduction of genes for enzymes that degrade and detoxify the herbicides. One example in this category is bromoxynil in which resistance was engineered through the introduction of a bacterial gene that codes for an enzyme that converts bromoxynil to an inactive metabolite. Another example is glufosinate (phosphinothricin, Liberty). Resistance to this herbicide can be engineered using a bacterial gene that acetylates and inactivates the herbicide. The last example is 2,4-D, a plant hormone that causes arrested development. Again, genetic engineering strategies employ a bacterial gene that encodes an enzyme that degrades 2,4-D. Cotton has been engineered to be resistant to this herbicide in order to prevent crop loss due to spray drift.

Although progress in engineering herbicide-resistant crops has been rapid, there are some problems with this approach. Technical problems include finding an effective strategy for engineering resistance and identifying the target site of the herbicide or a degradative or inactivating factor. In addition the resistance factor must be engineered to be expressed in the proper place and in sufficient levels.

The herbicides for which tolerance is being engineered are generally low-use rate, low-toxicity, rapid-turnover herbicides. Despite this, there are some environmental issues which, although not unique to genetically engineered varieties, must be taken into consideration.

Generation of herbicide-tolerant weeds due to monoherbicide application. Frequent and long-term application of a single herbicide becomes more likely because of the use of specific herbicide-tolerant varieties. The tendency of the industry to engineer a large number of crop species with tolerance to a single herbicide will create pressure for this to occur

Transfer of resistance genes to related weeds. Many crops in the U.S. in which tolerance is being developed do not have weedy relatives here; exceptions are sugarbeet, certain cucurbits, radish, canola, oats, and carrots. As seed is moved internationally, however, the possibility of outcrossing with wild relatives in centers of diversity will increase; control measures should be instituted. In general, however, unless pollen travels over long distances to pollinate weeds located far away, this problem will be minimal.

Encroachment of weeds not affected by the herbicide, i.e. weed shift. Development of elite germplasm resistant to a single herbicide, without comparable varieties resistant to other herbicides with dissimilar modes of action, could lead to an exacerbation of the weed shift problem.

Insect resistance. A now standard method for engineering insect resistance in crop species is the use of the toxin genes from Bacillus thuringiensis; this is the basis for Bollgard cotton and the commercially available maize that is European corn borer-resistant. These toxins are activated by cleavage in the midgut of insects; however, making engineered toxins that are effective against certain insects has not been straightforward. For example for a certain insect pest of alfalfa, Spodoptera armyworm, the situation was not straightforward. It required some extensive in vitro testing of synthetic toxins. In the end a specific synthetic delta-endotoxin gene was identified, which when expressed in optimum amounts in alfalfa, shows evidence of protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). In other cases new types of Bt's are being discovered and also some insecticidal compounds from other microbial species.

Viral resistance. Infection with viruses is a problem with many crop species; however the sources of natural resistance are limited and chemical protection is not effective in many instances. A standard method for engineering viral resistance is the use of the coat protein gene. This approach has been used successfully for many viruses in many different plant species and the Freedom II squash was released in the marketplace last year. In the coming year genetically engineered papaya will enter the marketplace. This crop results from efforts to protect it against papaya ringspot virus, which had devastated certain production areas in Hawaii. Because there are limitations to the coat protein approach in terms of uniform applicability and broad protection to related viruses, it is necessary to develop alternative strategies that complement or replace the coat protein strategy. These have included the use of viral polymerase and movement protein genes; such strategies require an in-depth understanding of the architecture of the

Fungal and bacterial resistance. Methods for protecting against fungal pathogens involve the identification of natural plant protection compounds that, when expressed in higher amounts in appropriate tissues of the host plant, can lessen fungal damage. These are sometimes compounds deriving from secondary metabolic pathways. Work on compounds, such as these, can lead to approaches where increased or broader-spectrum disease resistance can result from modifying expression levels or timing of unmodified secondary metabolite (e.g. phytoalexin) or modifying the phytoalexins themselves to create new phytoalexins with novel activities.

In addition natural defense genes have been isolated for resistance to various pathogens in different plant species Of the first three such genes that were isolated, it was discovered that they share common domains. Understanding these domains and their function, it will be possible to devise plant protection strategies using these genes that provides broad spectrum resistance against a variety of pathogens.

Nematode resistance. A variety of approaches have been utilized to protect against damage from nematodes. Regulatory regions have been identified which are uniquely engaged when the nematode burrows into the root; simple physical damage of the root is not sufficient to turn on the promoter. This regulatory region can then be hooked to nematicidal proteins, which can then be delivered to the nematode at its precise site of attack. Such nematicidal proteins include several different proteinase inhibitors and lectins, which act in some cases synergitically to interfere with the sexual development of the nematode.

In addition, recently a natural resistance gene was isolated from tomato and found to provide protection in engineered plants to not only nematodes but also aphids, which have an entirely different mode of attack and damage pathway. It was found, after comparison of the gene to other natural resistance genes (fungal, viral and bacterial) from other species, that the nematode resistance gene shared common domains with these other genes, again pointing to the potential universality of these mechanisms and opening the door for creating some engineered proteins with widespread efficacy.

Harvest and Post-harvest Characteristics

Another arena in which we have seen product enter the marketplace and in which much research is being conducted relates to harvest and post-harvest characteristics. Two different approaches to delayed ripening have been commercialized. The approach of the Flavr Savr tomato utilized a technique allowing growers and producers extra time to pick and deliver tomatoes to the marketplace by interfering with polygalacturonase synthesis, an enzyme involved in pectin deposition. In this case the ripening process was delayed by several days but was not halted. In another approach to interfering with the ripening process, researchers stopped the ripening process completely by interfering with the production of ethylene, which is essential to ripening. By this process it is possible to leave a tomato on the vine for up to 100 days past the time when it would ordinarily be ripe and, once it is picked, to ripen the tomato on demand by exposure to ethylene. This formed the basis for the Endless Summer tomato, which was on t

Another post-harvest characteristic, which is the focus of genetic engineering technologies is the achievement of more uniform ripening. This was first accomplished with the cherry tomato, but the basic approach is also being attempted with processing tomatoes in order to achieve less waste and aid harvesting. This approach involves the manipulation of genes involved in slowing the degradation of ethylene.

Agronomic Traits

Nitrogen fixation and utilization. Fundamental work on the process of nitrogen fixation has been pursued for decades. With the advent of molecular techniques, pace of that research has increased and has broadened to include fundamental studies on the nature of the plant-microbe interaction necessary for nodule formation and nitrogen fixation. Work on the bacterium itself has resulted in engineered Rhizobium strains that have increased rates of nitrogen fixation in limited field trials, but those strains have not yet been released for widespread use by the EPA. Studies focused on understanding and manipulating the host range of nitrogen-fixing bacteria are also underway with the expectation of being able to extend the range of crop species that can have productive commensal relationships with bacterial nitrogen fixers.

In addition to looking at improving the efficiency of nitrogen fixation, fundamental work aimed at understanding nitrogen fertilizer use efficiency is also underway. This work hopes to lead to engineered plants, e.g. wheat and corn, that more efficiently use applied fertilizers.

Male sterility

The development of hybrid varieties that result in increased yields of crop species has been a tedious process, requiring in many cases the manual detasseling of the male parent to prevent self-fertilization. Large scale production of hybrid seed, however, required more efficient mechanisms to suppress pollen production. Classical geneticists have identified naturally occurring male-sterile lines of some crops that were used for commercial seed production. Such strategies also required the identification of "restorer lines" that could restore male fertility by counteracting the genes that cause male-sterility. This is more easily accomplished with some crops than with others.

With the advent of genetic engineering, it became possible to develop strategies to create male sterile plants. These strategies made use of the fact that one could identify regulatory sequences that would lead to very specific tissue expression. In one strategy a promoter was identified that only drove expression in the inner layer of the pollen sac, the tapetum. Linked to this promoter was a bacterial enzyme, which degrades RNA, thereby interrupting the development pollen grains. A restorer of fertility was also identified; this protein is a chemical coupler of the RNA-degrading enzyme; the tight coupling of these two proteins inactivates the RNA-degrading enzyme, thus restoring fertility. This approach was used in canola first and is now being attempted in a variety of different crop and vegetable species.

Technology Protection System or "Terminator Technology"

This approach is a relatively new one patented by a small cotton company, Delta and Pine Land, and the USDA; the Monsanto Corporation has made a bid to buy DP&L, but that deal is still awaiting approval by the Department of Justice. The fundamental idea with this technology is to prevent a plant from reproducing itself by interfering with seed development, thus insuring that the user of the seed cannot replant a field without repurchasing the seed, thereby protecting the company's investment in developing the variety.

The system works through the interplay of three introduced genes. One of these genes leads to the production of a toxin that kills the seed in the final stage of development. This gene is initially interrupted by a stretch of DNA between the toxin gene itself and the regulatory region or promoter that is responsible for its synthesis. This gene configuration can then be transplanted or crossed with another genetically engineered plant that produces a scissor-like enzyme, capable of removing the segment in the toxin gene, thereby activating it and causing the seed to die. The third component of the system is a repressor gene that prevents the synthesis of the scissor-like enzyme. If all three components are present, the terminator technology is dormant and the seed is fertile. If the seeds are then treated with a stimulator, in one case an antibiotic called tetracycline, this compound can then block repressor of the scissor-like protein and this gene product can then snip out the DNA that is preventing the syn

DP&L is currently pursuing this technique in cotton and wheat and expect these products to be on the market by 2005. With the entry of Monsanto into the equation, application of the technology could accelerate into many more crops and the advent into the marketplace could be much more rapid. There has been much furor and debate about this technology since obviously it can be viewed as an interference with farmer's rights and insures that farmers return every year to purchase a new lot of seeds. This has led a large, international, not-for-profit agricultural research organization, the Consultative Group on International Agricultural Research, or CGIAR, to consider banning the use of the technology in crop development programs.

While certainly there is a negative aspect to the technology related to farmer's rights, I would offer a counter perspective on the use of such a technology. Another concern held by environmentalist and certain agriculturalists is that certain traits, like herbicide tolerance and certain other traits, will escape from the crop plant and enter into wild species located nearby, thereby creating situation where it is difficult to control the wild species or it has some other new, adverse characteristic. This issue has been raised with, for example, sorghum and Johnson grass, oat and wild oat, rice and red rice, canola and its wild Brassica relatives. The use of such a technology for engineering plants that have wild relatives nearby is a prudent approach to preventing the dissemination of genes to wild populations.

Value-added products

There is another area that does not involve plant growth regulators but is being intensely pursued by both the public and private sectors. This relates to the improvement of existing plant products, ones that already exist but for which new or improved functions can be identified that increase the value of the product. An example would be specialty oils from rape or soybean, which are designed to have improved heat stability. Another example would be the genetic engineering of sugarbeet to produce fructans, a sweet, no-calorie sugar substitute.

An intriguing application is in the area of functional foods or neutriceuticals. This line of foods is based on the fact that we can now identify in many cases natural products in foods that can either cause adverse human condition or prevent disease. Examples of those products focused on factors in foods that cause disease or undesirable pharmacological effects include ameliorating the allergenic potential of foods, such as wheat, milk and peanut or removing caffeine from coffee. In the category of natural products that prevent disease, there are many examples. Transgenic potatoes are being created which produce high quantities of GAD, an enzyme believed to be involved in the prevention of type II juvenile diabetes. Researchers have identified an element in grapes that increases blood flow in blood vessels; grapes could be engineered to increase the level of such an element. Several groups are coming up with margarine and oils, based on natural plant sterols and stanols, that are not only "low in choles

Summary

In summary, there are a number of areas relating to plant growth regulators that can be approached through genetic engineering that will likely impact agriculture in the coming decades. These range from engineered herbicide tolerance to male sterility, from improved pest resistance to terminator technologies and delayed senescence. While these technologies will certainly change agricultural practices, they will not be "magic bullets" that will eliminate the need for classical methods of plant production and protection. These technologies should be viewed as adjuncts to existing technologies that provide new tools for the farmer's and producer's tool box.